The present invention relates to coupling agents comprising a photolabile protecting group and to the use thereof for the functionalization of solid supports. The present invention also relates to the solid supports functionalized with these coupling agents, and also to the use thereof for the immobilization of biological molecules of interest, in particular of nucleic acid molecules.
Various methods for the covalent grafting of biological molecules of interest, and in particular of nucleic acid such as oligonucleotides (ON), onto a solid support have already been proposed. In the specific context of the attachment of ONs, these methods can be divided into two main categories:                1) In situ synthesis, which consists in constructing the ON molecule step by step on a solid support using the principle of synthesis via the phosphoramidite pathway. This method, described in particular in international application WO 97/39151, allows the preparation of surfaces with a high density of ONs. In addition, by means of a set of blanking plates and the use of photolabile protecting groups, it allows the targeting of the various ONs on the support. However, this method has a certain number of major disadvantages. In particular, the ONs attached to the support cannot be characterized and it is very difficult to incorporate ONs carrying modifications.        2) Synthesis by deposition, or ex situ, which consists, firstly, in preparing the ON conventionally and then, secondly, in attaching it to a solid support using a suitable chemical reaction. This second method has in particular the advantage of using ON molecules that can be characterized before they are attached to the solid support.        
The invention which will be described hereinafter falls more especially within the context of methods of synthesis by deposition.
Several types of methods of synthesis by deposition have already been described in the prior art, in particular as regards the various techniques for localizing the grafting of the ON.
Mention may, firstly, be made of methods using the electrochemical pathway, which consist in anchoring the ON, functionalized beforehand with an electrically conducting group, for instance a pyrrole group, via electropolymerization of the pyrrole residue. This type of electrochemical ON deposition method has in particular been described in patent FR 2 703 359. It nevertheless has the drawback of requiring the use of an electrically conducting support, which makes the final component complex. Furthermore, in a component of “Lab on a Chip” type, the various fabrication steps can impair the quality of the electrodes and therefore their functionality.
Mention may, secondly, be made of the photochemical pathway, which appears to be more promising since the management of the surfaces and of the chemical solutions to be used is externalized. Two approaches have been developed:                i) direct photografting between the surface and the target molecule. In this case, the photografting has the disadvantage of involving free-radical reactions which are not very selective;        ii) the freeing of a function protected with a photolabile protecting group on the support, so as to allow subsequent reaction with the target ON in order to produce attachment thereof. In this case, the supports used are generally functionalized with coupling agents which are bifunctional compounds comprising, at one of their ends, a function for attachment to the surface of the support and, at the other end, a function that is reactive with respect to a target molecule comprising a complementary chemical function, said reactive function of the coupling agent being protected with a photolabile group. However, in order to be competitive, the latter approach must satisfy a certain number of criteria. These criteria are the following:        the reaction between the activated support and the target molecule must be rapid; this reaction can only occur when the reactive functions of the coupling agent have been freed from their protecting group (activation reaction);        the yield from this reaction must be high;        the method of synthesis for preparing the coupling agents must be simple to carry out and comprise as few steps as possible;        the use of mild reaction conditions (water, for example);        the bond formed between the coupling agent and the target molecule must be stable under various conditions of temperature and pH so as to allow very flexible use of the support thus functionalized.        
Now, it appears that the various methods proposed to date do not satisfy all these criteria entirely satisfactorily, in particular due to the nature of the various coupling agents used.